1. Field of the Invention
This invention pertains to the field of high throughput assays for identifying modulators of interactions between the signal transducer and activator of transcription molecules STAT4 and STAT6 and their corresponding receptors.
2. Background
The growth, differentiation, and functional responses of many cells are controlled by cytokines, growth factors, and hormones. While many of these responses are desirable for normal development of an organism, or to help an organism fight an attacking pathogenic organism or a disease condition, in other situations the cellular response to a given stimulus is inappropriate. For example, T helper cells are an integral part of an organism""s defense against harmful antigens. However, when produced at art inappropriate time, T helper cells can attack the organism itself, thus resulting in an autoimmune disease. In other situations, it may be desirable to trigger or enhance a particular response, even in the absence of the usual stimulatory signal.
Development of methods by which one can influence the response of a cell to a given cytokine or growth factor stimulus would be facilitated by an understanding of the signal transduction pathway by which an interaction between a cell and a cytokine is translated into a response by the cell. Generally, cytokines bind to a cell surface receptor of the cytokine receptor superfamily. Binding of a cytokine to its corresponding receptor causes receptor aggregation as well as association of a Janus protein tyrosine kinase (Jaks) with the receptor. Receptor aggregation allows the Jaks to transphosphorylate each other, which dramatically increases their catalytic activity. The activated Jaks then phosphorylate the receptor. Also phosphorylated by the activated Jaks is a polypeptide that is responsible for transmitting the signal to the cell""s nucleus. These polypeptides are members of a family of proteins known as Signal transducers and activators of transcription (STAT; Darnell et al. (1994) Science 264: 1415; for review, see, e.g., Ihle et al. (1994) Trends Biochem. Sci. 19: 222; Ihle et al. (1995) Trends Genetics 11: 69). STATs are activated by contact with the phosphorylated receptor; activation results in the STAT polypeptides forming a dimer and entering the nucleus, where the STAT dimer binds to the regulatory region of a gene that is inducible by the particular cytokine. Binding of the activated STAT dimer triggers transcription of the gene.
Seven STAT polypeptides are known (STAT1, STAT2, STAT4, STAT5a, STAT5b, and STAT6); these polypeptides have molecular masses from 84-113 kDa. Each STAT protein contains a Src homology-2 (SH2) domain capable of recognizing one or more phosphotyrosine sequences in the cytoplasmic portion of the activated receptor (Shuai et al. (1993) Nature 366: 580). Each cytokine receptor is specific for a particular STAT protein, and each STAT activates transcription of certain genes, thus providing two layers of cytokine specificity.
At least two of the STAT polypeptides, STAT4 and STAT6, are intimately involved in regulation of immune responses. STAT4 transduces to the nucleus signals from the IL-12 receptor. IL-12 is involved in the development of a TH1 immune response (Kaplan et al. (1996) Nature 382: 174-177), which is part of an organism""s defense against intracellular pathogens. IL-12 is also necessary for the T-cell-independent induction of the cytokine interferon (IFN)-xcex3, which is a key step in the initial suppression of bacterial and parasitic infections. Knockout mice which lack STAT4 were found to be defective in all IL-12 functions tested, including the induction of IFN-gamma, mitogenesis, enhancement of natural killer cytolytic function and TH1 differentiation (Thierfelder et al. (1996) Nature 382: 171-174).
IL-4 signals are transduced to the nucleus by STAT6. IL-4 is a key cytokine in the initiation of a TH2 immune response, and also activates B and T lymphocytes. STAT6-deficient mice were deficient in IL-4 activities (Kaplan et al. (1996) Immunity 4: 313-319; Takeda et al. (1996) Nature 380: 627-630; Shimoda et al. (1996) Nature 380: 630-633).
Because of the importance of STAT4 and STAT6 in modulating the immune response of an organism, both in response to infection and in undesirable conditions such as inflammation, allergic reactions, and autoimmune diseases, a need exists by which the clinician can enhance or reduce STAT4 and STAT6 signals. Intervention at the STAT level would have significant advantages compared to previous approaches, which typically target the IL-4 or IL-12 cytokine itself, or the interaction of the cytokine with the receptor. Disruption of cytokine function itself can cause a variety of undesirable side effects. These can be avoided by intervening at the level of STAT-mediated signal transduction. However, identification of agents that can modulate STAT4 and STAT6-mediated signal transduction has been hampered by the lack of suitable assays. Assay of binding of STAT4 and STAT6 to their corresponding receptors, and identification of agents which increase or decrease the degree of such binding, has been difficult because of the high rate at which the STAT4 and STAT6 polypeptides bind to (kon) and leave the receptor (koff) after initial binding. The present invention fulfils this need for effective assays by which modulators of STAT4 and STAT6 binding can be identified, as well as other needs.
The invention provides receptor peptides that have a high affinity for STAT6 polypeptides, and thus are useful in assays to identify modulators of STAT6 binding to corresponding receptors. The peptides include an amino acid sequence YX1X2X3 (SEQ ID NO:1), wherein X1 is selected from the group consisting of K, V, R, I, M, a nonnatural amino acid such as tert-butyl glycine, norvaline, cyclohexylalanine, or allothreonine; X2 is selected from the group consisting of P, A and S, and X3 is selected from the group consisting of W, Y, F, H, L and an aromatic normatural amino acid. Suitable aromatic nonnatural amino acids include, for example, p-iodophenylalanine, 1-napthylalanine, benzothiophenylalanine, 3-iodotyrosine, p-chlorophenylalanine, m-trifluoromethylphenylalanine, and o-chlorophenylalanine. In preferred embodiments, the peptide does not include the amino acid sequence YKPF (SEQ ID NO:2). Also provided are STAT6 receptor peptides of the invention in which the tyrosine is phosphorylated; such phosphorylated receptor peptides bind to STAT6 with high affinity.
In another embodiment, the invention provides receptor peptides that have a high affinity for STAT4 polypeptides. These peptides include an amino acid sequence GYLPZ3NID (SEQ ID NO:3), wherein Z3 is selected from the group consisting of Q, H, N, and W; other STAT4 receptor peptides include the amino acid sequence GYDMPHVL (SEQ ID NO:4). Peptides having the tyrosine phosphorylated are also provided, these STAT4 receptor peptides are capable of binding to STAT4 with high affinity.
Another embodiment of the invention provides methods of screening for modulators of STAT6 binding to a STAT6 receptor. The methods involve incubating a reaction mixture comprising a STAT6 polypeptide, a potential binding modulator, and a STAT6 receptor peptide as set forth herein. The amount of binding of the STAT6 polypeptide to the receptor polypeptide is determined and is typically compared to the amount of binding observed in an assay which lacks the potential binding modulator in order to determine whether the potential binding modulator increases or decreases binding of the STAT6 polypeptide to the receptor peptide.
The invention also provides methods of screening for compounds that modulate STAT4 binding to a STAT6 receptor. These methods involve incubating a reaction mixture comprising a STAT4 polypeptide, a potential binding modulator, and a receptor peptide which comprises an amino acid sequence GYDMPHVL (SEQ ID NO:4) or GYLPZ3NID (SEQ ID NO:3), wherein the tyrosine is phosphorylated and Z3 is selected from the group consisting of Q, H, N, and W. One then determines whether the binding of the STAT4 polypeptide to the receptor peptide is increased or decreased in comparison to an assay which lacks the potential binding modulator.